Beta-Ketoacyl ACP synthase III (KASIII) catalyzes the decarboxylative condensation of acetyl CoA and malonyl ACP to generate beta-ketobutyryl ACP. This reaction represents first step in initiation of fatty acid biosynthesis in the dissociated type II fatty acid synthases (FASs) of plants and bacteria. Thiolactomycin (TLM) is a potent antibiotic that has been shown to efficiently inhibit this enzyme in a broad range of organisms, including pathogenic bacteria. This application seeks to discover novel inhibitors of KASIII as potential agents to treat emerging and reemerging human pathogens. An approach that draws both on the proven track record of natural products as sources of bioactive molecules and the powerful techniques of structure-based drug discovery will be taken. In the first specific aim of the project an enzyme-coupled spectrophotometric assay for KASIII will be established. This assay will then be used to screen a library of 2000 purified diverse natural products that have previously been structurally characterized. Two additional libraries, consisting 3000 crude natural product extracts, and 3000 pure compounds prepared by multiple parallel synthetic approaches using natural products as templates, will be screened for KASIII inhibitors. The second specific aim of the project is to determine conditions in which KASIII can be crystallized, preferably with either TLM, or malonyl ACP in the active site. X-ray diffraction will then be used to obtain the 3-dimensional structure of KASIII. In the long term this information will be used as a basis to rationally design KASIII inhibitors.